No presente trabalho foram avaliados em ratos adultos os efeitos da desnutrição precoce e/ou de inibidor seletivo de recaptação de serotonina sobre o consumo alimentar e agressividade intraespecífica. Outrossim, foram estudadas as inter-relações entre desnutrição pregressa, agressividade e parâmetros da resposta imune. Desta pesquisa, foram originados cinco artigos científicos. Destes, dois foram publicados, dois aceitos e um está submetido. Todos os artigos serão apresentados aqui em ordem cronológica e em suas versões originais.
5.1. Primeiro artigo, intitulado: “ACTION OF SELECTIVE SEROTONIN
REUPTAKE INHIBITOR ON AGGRESSIVE BEHAVIOR IN ADULT RAT SUBMITTED TO THE NEONATAL MALNUTRITION”. Publicado como artigo
original na revista: Arq Neuropsiquiatr, 59(3-A):499 – 503, 2001.
Neste artigo, foram estudados os efeitos da desnutrição durante o aleitamento sobre a agressividade intraespecífica em ratos adultos tratados ou não com inibidor seletivo de recaptação de serotonina. Neste estudo foi observado que a desnutrição durante o período de desenvolvimento rápido do cérebro tornou os ratos adultos resistentes aos efeitos anti-agressividade do citalopram.
5.2. Segundo artigo, intitulado: “EARLY MALNOURISHED RATS ARE NOT
AFFECTED BY ANOREXIA INDUCED BY A SELECTIVE SEROTONIN REUPTAKE INHIBITOR IN THE ADULT LIFE” Publicado como artigo original
na revista: Nutritional Neuroscience, 5(3):211–214, 2002.
Em seqüência ao estudo apresentado no primeiro artigo, referente a influencia da desnutrição sobre o sistema serotoninérgico, o presente trabalho teve como principal objetivo investigar os efeitos da desnutrição durante o aleitamento sobre o consumo alimentar em ratos adultos tratados ou não com citalopram. Neste estudo foi observado que a desnutrição durante o período de desenvolvimento rápido do cérebro alterou a anorexia induzida por citalopram em ratos adultos.
5.3. Terceiro artigo, intitulado: “MALNUTRITION DURING BRAIN GROWTH
SPURT ALTERS THE EFFECT OF FLUOXETINE ON AGGRESSIVE BEHAVIOR IN ADULT RATS”. Aceito para publicação na revista: Nutritional Neuroscience, 2003.
Ainda, dando continuidade aos estudos referentes a influência da desnutrição sobre o sistema serotoninérgico, o presente trabalho teve como principal objetivo investigar os efeitos da fluoxetina (outro ISRS) sobre a resposta agressiva em ratos adultos submetidos à desnutrição durante o período de rápido desenvolvimento do cérebro. Neste estudo, nós observamos uma hiporresponsividade a fluoxetina em ratos adultos submetidos à desnutrição precoce.
Running Title: Malnutrition, serotonin and aggressive behavior
Malnutrition during Brain Growth Spurt Alters the Effect of Fluoxetine on Aggressive Behavior in Adult Rats.
J. M. BARRETO-MEDEIROSa, E. G. FEITOZAa, K. MAGALHÃESa, J.E. CABRAL-FILHOb, F.M. MANHÃES-DE-CASTROa , C. M. M. B. DE-CASTROc , R. MANHÃES-DE-CASTROa *
a
Departamento de Nutrição, Universidade Federal de Pernambuco (UFPE), Recife, PE, Brazil; bInstituto Materno Infantil de Pernambuco, Recife, PE, Brazil; cLaboratório de Imunopatologia Keizo Asami, Universidade Federal de Pernambuco, Recife, PE, Brazil.
KEY WORDS: aggressive behavior, malnutrition, selective serotonin reuptake inhibitor.
*Corresponding author: Departamento de Nutrição, Universidade Federal de Pernambuco, Cidade Universitária, 50670-901,Recife, PE, Brasil. Tel.: (081) 271.8471. Fax: (081) 271.8473. E-mail: rcastro@nutricao.ufpe.br.
The effect of the malnutrition during suckling period on the aggressive behavior was investigated in adult rats treated or not with fluoxetine, a selective serotonin reuptake inhibitor. Sixty-four Wistar male rats were allocated in two groups, according to their mother’s diet during lactation. The well-nourished group was fed by mothers receiving a 23% protein diet; the malnourished one by mothers receiving a 8% protein diet. After weaning, all rats received the 23% protein diet. On the 90th day after birth, each nutritional group was divided in two subgroups, which received respectively a single daily injection of fluoxetine (10 mg/kg) or saline (0.9% NaCl) for 14 days. The treatment with fluoxetine reduced the aggressive response in well-nourished but not in malnourished rats. These findings suggest that the serotoninergic system has suffered consequences of the malnutrition during the critical period of brain development, persisting even after a long period of nutritional recovery.
The growth of the central nervous system and its developmental processes (gliogenesis, neuronal differentiation, migration, synaptogenesis, etc) occur with great intensity during the suckling period in the rat (Morgane et al., 1993). This period is called “brain growth spurt” (Dobbing, 1968) and in it the brain is highly vulnerable to several kinds of challenges. Nutritional insults (Morgane et al., 1992) or pharmacological manipulations early in life (Manhães de Castro et al., 1993) can cause irreversible alterations.
The effects of malnutrition on neurotransmitters systems, in particular the serotoninergic one, has deserved special attention, since this system plays a role in several functions, such as pain sensitivity, sleep control, mood, sexual behavior, feeding control and aggressiveness (Chopin et al., 1994).
The role of serotonin in the control of aggressive behavior has been demonstrated through the use of pharmacological instruments (Manhães de Castro et al., 2001). On the other hand, there are drugs that act as selective serotonin reuptake inhibitors increase serotonin availability in synapses and, consequently, the action of this neurotransmitter (Sánchez and Meier, 1997). Fluoxetine, a potent serotonin reuptake inhibitor(Hyttel, 1994; Sánchez and Hyttel, 1999), is one of those substances.
Mounting evidences indicate that fluoxetine decreases aggressive behavior in various animal species and models of aggression (Datla et al., 1991; Sánchez and Hyttel, 1994; Sperry et a., 2003). However, to our knowledge, there are no studies on the effects of fluoxetine in the aggressive behavior of adult rats malnourished early in life. The present work aimed to investigate the effect of fluoxetine on aggressiveness of adult rats submitted to malnutrition during the brain growth spurt.
the offsprings were housed in polyethylene cages in litters of 6 pups, randomly distributed to each mother. They were then allocated in two nutritional groups according to the mother diet during lactation: a well-nourished group (n=32) fed by mothers receiving a 23% protein diet (Purina chow) and a malnourished group (n=32) fed by mothers receiving a 8% protein diet. Following weaning (on the 24th day of age), all rats were fed with the 23% protein diet ad libitum. Body weights were determined on the 1st, 24th and 90th day. On the 90th day after birth, the well- nourished animals and the malnourished ones were divided according to the four following treatments (n=16, each one): well-nourished plus saline; well-nourished plus fluoxetine; malnourished plus saline; malnourished plus fluoxetine. Single injections of fluoxetine (10 mg/kg, i.p., 1 ml/100mg, dissolved in saline) or saline (0.9% NaCl) were administered during 14 days. Twenty-four hours following this treatment period the animals were submitted to aggressiveness tests. The tests were accomplished in an acoustic isolated room, by using a box (20 x 20 x 20 cm) with one the walls transparent and the floor formed by parallel metallic bars (inter-bars distance: 1.3cm), connected to an electric scrambled current source. The test consisted of placing a pair of rats of the same treatment group (matched by weight) in the box, where they received a session of stimuli to induce fight responses. Each stimulus (an electric foot- shock) was produced by a 1,6 mA - 2 s current pulse. Each session lasted 20 min being composed by 5 stimuli separated by a 4 min interval. During the first 3 min of this interval, the duration of the aggressive responses was measured by using a digital chronometer. So the total time for observation of aggressive behavior was 900s. In the last minute of each interval, annotations and verification of the equipment were proceeded. The aggressive response was defined as the presentation of, at least, one of the two following behaviors: a) the animals stayed lifted up on the hind paws, facing
one to the other, in a threatening posture but without direct contact, or b) the animals maintained evident physical contact (besides being scratched, exhibition of the teeth and emission of characteristic vocalization). For statistical comparisons, data were previously tested for normality (Kolmogorov-Smirnov test) and variance homogeneity (Levene median test). Body weights passed (p>0.05) in both criteria, but aggressiveness, not (variance homogeneity, p<0.05). So body weights (between nutritional groups) were compared by Student t-test, and aggressiveness by Kruskal- Wallis, one way analysis of variance followed by Dunn's test for multiple comparisons. The null hypothesis was rejected when p≤0.05.
Compared to well-nourished animals, the malnourished ones suffered a reduction of the body weight (p<0.05) both in suckling and in adult periods (table I). Concerning the aggressive behavior (figure 1), observed after pharmacological manipulation, the fluoxetine groups showed a reduction of aggressiveness as compared to the well- nourished saline group (Dunn test, p<0.05). However, there was no significant difference between malnourished saline and malnourished fluoxetine animals.
The results of the present study showed that malnutrition during the critical period of brain development induces a persistent body weight deficit in rats. The data corroborate previous experimental evidence showing lower body weight of animals submitted to malnutrition during the suckling period (Sobokta et al., 1974). Therefore, body weight reduction caused by the maternal low protein diet attests to the efficiency of the employed malnutrition model.
The inhibitory effect of fluoxetine on aggressive behavior observed by others (Datla
brain induced by fluoxetine (Baumann and Rochat, 1995). This is a plausible hypothesis since an anti--aggressiveness action of serotonin in humans beings (Coccaro and Kavoussi, 1997) and in animals (Sánchez and Hyttel, 1994; Sperry, et
al., 2003) has been demonstrated. In contrast, an increased aggressiveness has been
observed after diminished brain serotonin (Kyes, 1993).
Although fluoxetine seems to enhance not only serotonin availability but that of dopamine and norepinephrine as well (Hyttel, 1994; Wong et al., 1995), the participation of these catecholamines in the reduction of aggressive behavior can be ruled out. In fact, evidences indicate that dopamine and norepinephrine facilitate and do not inhibit the aggressiveness (Eichelman, 1990; Datla et al., 1991; Pruus et al., 2000).
Since fluoxetine has also an anxiolytic effect (Sánchez and Meier 1997), the possibility exists that hipoaggressiveness here observed would be associated to a reduced anxiety of the animals. This influence however could be little because the anxiolytic effect of fluoxetine is weak (De Vry et al., 1993)
The reduced response to the fluoxetine of malnourished animals as compared to those malnourished that did not receive the drug is a major subject of this study and is noteworthy. In rats, the first serotoninergic neurons appear between the 12° and the 14° gestation day (Lauder and Bloom, 1974) but the final density and definitive location of terminals are established during the postnatal maturation of the central nervous system (Lidov and Molliver, 1982). Therefore, the malnutrition imposed early in life could cause alterations in the serotonin neurotransmission system, reflecting on its functional responses to drugs. Medeiros et al. (2001) showed that adult rats malnourished during suckling are hypo-responsive to the inhibitory effect of citalopram -another selective serotonin reuptake inhibitor- on the intraspecific aggressive behavior. Barreto-
Medeiros et al. (2002) also observed that early malnourished rats are not affected in adult life by anorexia induced by citalopram.
We cannot rule out the possibility of an anti-aggressive response of malnourished rats to other fluoxetine doses. However the similar responsiveness between malnourished saline and malnourished fluoxetine groups, here observed, allows us suppose that the anti-aggressiveness effect of fluoxetine does not occur, at least, by chronic administration of a 10mg dose.
Furthermore, as long ago as the eighties, Hall et al. (1983) showed that early protein malnourished rats stimulated by the serotonin agonist N, N-dimethyltryptamine presented a lower performance than well-nourished animals in several behavioral tests, such as the serotoninergic syndrome, the rota-rod and the treadmill. This lower responsiveness was supposed by these authors to be associated to serotonin neuronal processes and to alterations of brain serotonin due to malnutrition (Hall et al., 1983).
On the basis of this work we can suggest that, the hypo-responsiveness to fluoxetine of rats submitted to protein restriction during the brain growth spurt and then nutritionally recovered, is due to that nutritional deficit. Therefore the nutritional insult early in life could to have a long-lasting effect upon the functioning of the serotoninergic system.
Acknowledgements
The authors thank Lúcia Maria Pires Ferreira for her superb and timely assistance. Jairza Maria Barreto Medeiros is the recipient of a fellowship from the CAPES/PICDT/UFBA.
affected by anorexia induced by a selective serotonin reuptake inhibitor in adult life”, Nutritional Neuroscience 5, 211-214.
Bauman, P. and Rochat, B. (1995) “Comparative pharmacokinetics of selective serotonin reuptake inhibitors: a look behind the mirror”, International Clinical
Psychopharmacology 10,15-21.
Chopin, P., Moret, C. and Briley, M. (1994) “Neuropharmacology of 5- hydroxytryptamine1B/1D receptor ligands”, Pharmacology and Therapeutics
62,385-405.
Coccaro, E.F. and Kavoussi, R.J. (1997) “Fluoxetine and impulsive aggressive behavior in personality-disordered subjects” Archives General Psychiatry
54,1081-1088.
Datla, K.P., Mitra, S.K., Bhattacharya, S.K. (1991) “Serotonergic modulation of footshock induced aggression in paired rats” Indian Journal of Experimental Biology 29, 631-635.
De Vry, J., Benz, U., Schreiber, R., Traber, J. (1993) “Shock-induced ultrasonic vocalization in young adult rats: a model for testing putative anti-anxiety drugs”,
European Journal of Pharmacology 249, 331-339.
Dobbing, J.(1968) “Vulnerable periods in developing brain”. In: Davison, A.N. and Dobbing, J., eds, Applied Neurochemistry (Blackwell, Oxford), pp 287-316.
Eichelman, B.S. (1990) “Neurochemical and psychopharmacologic aspects of aggressive behavior”, Annual Review of Medicine 41, 149-158.
Fuller, R.W. (1996) “Fluoxetine effects on serotonin function and aggressive behavior”, Annals of the New York Academy of Sciences 20, 90-97.
Hall, R.D., Leahi, J.P. and Robertson, W.M. (1983) “Hyposensitivity to serotoninergic stimulation in protein malnourished rats”, Physiology and Behavior 31, 187-195. Hyttel, J. (1994) “Pharmacological characterization of selective serotonin reuptake
inhibitors (SSRIs)”. International Clinical Psychopharmacology 9 (suppl. 1), 19- 26.
Kyes, G.B. (1993) “Aggression and brain serotoninergic responsivity: response to slides in male macaques”, Physiology and Behavior 2, 205-208.
Lauder, J.M. and Bloom, F.E. (1974) “Ontogeny of monoamine neurons in locus coeruleous, raphe nuclei and substantia nigra of the rats 1, Cell differentiation”,
Journal of Comparative Neurology 155, 469-481.
Lidov, H.G.W. and Molliver, M.E. (1982) “A immunohistochemical study of serotonin neuron development in the rat: ascending pathways and terminal fields”, Brain
Research Bulletin 8, 389-416.
Manhäes-de-Castro, R., Barreto Medeiros, J. M., Mendes da Silva, C., Ferreira, L.M.P., Guedes, R.C. A., Cabral Filho, J.E. and Costa, J.A. (2001) “Reduction of
intraespecific aggression in adult rats by neonatal treatment with a seletive serotonin reuptake inhibitor”, Brazilian Journal of Medical and Biological
Research 34, 121-124.
Manhäes-de-Castro, R., Cabral Filho, J.E., Costa, J. A., Costa, F.B.R., Galindo, M.A.C. and Hecksher, C.A. (1993)“ Neonatal treatment with naloxone causes permanent hyperalgesia in rats”, Brazilian Journal of Medical and Biological
Research 26, 747-751. .
Medeiros, J.M., Mendes da Silva, C., Sougey, E.B., Costa, J. A., De Castro, C.M.M.B. and Manhães de Castro, R. (2001) “Action of selective serotonin reuptake inhibitor on aggressive behavior in adult rats submitted to the neonatal malnutrition”, Arquivos de Neuro-psiquiatria 59 (3-A), 499-503.
Morgane, P.J., Austin-la France, R.J., Bronzino, J., Tonkiss, J. and Galler, J. R. (1992) “Malnutrition and developing central nervous system”, In: Isaacson, R.L. and Jensen, K.F., eds, The vulnerable brain and environmental risks. (Plenum Press, New York) Vol. 1, pp 2-42.
Morgane, P.J., Austin-la France, R.J., Bronzino, J., Tonkiss, J., Diaz-Cintra, S., Cintra, L., Kemper, T. and Galler, J. R. (1993) “Prenatal malnutrition and development of the brain”, Neuroscience and Biobehavioral Reviews 17, 91-128.
Pruus, K., Rudissaar, R., Skrebuhhova-Malmros, T., Allikmets, L., Matto, V. (2000) “Development of apomorphine-induced aggressive behavior: comparison of adult male and female Wistar rats” Methods and Findings in Experimental and Clinical
Pharmacology 22, 47-50.
Sánchez, C. and Hyttel, J. (1994) “Isolation-induced aggression in mice: effects of 5- hydroxytryptamine uptake inhibitors and involvement of postsynaptic 5-HT1A receptors”, European Journal of Pharmacology 264, 241-247.
Sánchez, C. and Meier, E. (1997) “Behavioral profiles of SSRIs in animal models of depression, anxiety and aggression”, Psychopharmacology 129:197-205.
Sánchez,C. and Hyttel, J. (1999) “Comparison of the effects of antidepressants and their metabolites on reuptake of biogenic amines and on receptor binding, Cellular
and Molecular Neurobiology 19: 467-489.
Sobokta, T. J., Cook, M. P. and Brodie, R. E (1974) “Neonatal malnutrition: neurochemical, hormonal and behavioral manifestations”, Brain Research 26:443- 457.
Sperry, T.S., Thompson, C.K. and Wingfield, J.C. (2003) “Effects of acute treatment with 8-OH-DPAT and fluoxetine on aggressive behaviour in male song sparrows (Melospiza melodia morphna)”, Journal of Neuroendocrinology 15, 150-160.
TABLE I. Body weight of well-nourished or malnourished rats during suckling.
Body Weight (g)
Experimental Groups 1st day 24th day 90th day
Well-nourished 7.5 ±0.15 49.0 ±0.85 314.2 ±9.83 Malnourished 7.3 ±0.11 23.8 ±1.07* 265.3 ±7.84*
Rats were fed by mothers receiving a 23% (Well-nourished group) or a 8% (malnourished group) protein diet during lactation. The animals were weighed on the 1st, 24th, and 90th day. The data are reported as mean (± SEM) of 16 animals. Comparisons between well-nourished and malnourished groups at the same age, were made by two-tailed Student t-test (* p<0.05).
FIGURE 1 725 - 406 598 - 217 895 - 238 719 - 122 0 200 400 600 800
Duration of agressive response (s)
WELL-NOURISHED SALINE WELL-NOURISHED FLUOXETINE MALNOURISHED SALINE MALNOURISHED FLUOXETINE
*
*
Figure Legends
Figure 1. Aggressive responses of fluoxetine or saline-treated adult rats, fed by mothers receiving a 23% or a 8% protein diet during lactation. Columns represent the medians of 16 animals; maximum and minimum values are inside columns. Comparisons among groups (Kruskal-Wallis test, p=0.01) and multiple comparisons between groups (Dunn's test,*p<0.05): well-nourished fluoxetine < well-nourished saline and malnourished fluoxetine < well-nourished saline.
5.4. Quarto artigo, intitulado: “THE EXPRESSION OF AN INTRASPECIFIC
AGGRESSIVE REACTION BEFORE A STRESSOR ALTERS THE IMMUNE RESPONSE IN RATS”. Artigo original aceito para publicação na revista: Brazilian Journal of Biology, 65 (3), 2005.
Nesse artigo, foi avaliado em ratos adultos se a expressão ou não da agressividade intraespecífica frente a um estressor pode alterar a resposta imune. Nesta pesquisa, foi observado que a expressão da agressividade intraespecífica parece ativar a resposta imune e potencializar a resposta humoral antígeno específica.
THE EXPRESSION OF AN INTRASPECIFIC AGGRESSIVE REACTION BEFORE A STRESSOR ALTERS THE IMMUNE RESPONSE IN RATS.
Jairza Maria Barreto-Medeiros1, Emmanuela Gomes Feitoza1, Kedma Magalhaes1, Rosângela R da Silva2, Francisco Machado Manhaes-de-Castro1, Raul Manhaes-de- Castro1, Célia MariaMachado Barbosa De-Castro2*
1
Departamento de Nutrição, Universidade Federal de Pernambuco, Cidade Universitária, 50670-901, Recife, PE, Brazil; 2Laboratório de Imunopatologia Keizo Asami, Universidade Federal de Pernambuco, Cidade Universitária, 50670-901, Recife, PE, Brazil.
Key words: intraspecific aggressiveness, leukocytes, humoral immune response.
Palavras-Chaves: agressividade intraespecífica, leucócitos, resposta imune humoral.
Running Title: intraspecific aggressive behavior and immune response
*
Correspondence to: Dra. Célia Maria Machado Barbosa de Castro. Laboratório de Imunopatologia Keizo Asami, UFPE, Recife, CEP 50670-901 PE, Brasil,Fax: 55 81 32718485
E-mail: ccastro@lika.ufpe.br
Number of figures: 04
ABSTRACT
The repercussion on the immune response of the expression of the intraspecific aggressiveness before a stressor was investigated in rats. To the 90 days of life, the animals were divided in three groups: control group (only the immunological measurements were accomplished); foot-shock (FS) (animals individually received FS) and intraspecific aggressive response (IAR) group (animals received FS and presented IAR). For immunological measurements, blood samples were collected immediately, 7 and 15 days after FS or IAR. FS reduced the total amount of leukocytes. However, the aggressiveness was accompanied, besides the reduction of the leukocytes number, by lymphocytes decrease and neutrophils increase. Moreover, an elevation in the leukocytes number associated to an increase in the humoral immune response was also observed one week after the IAR. In this study, the expression of the intraspecific aggressiveness before a stressor seems to activate the immune system and to potentiate the antigen specific humoral response.
RESUMO
A expressão de uma reação agressiva intraespecífica frente a um estressor altera
a resposta imune em ratos.
A repercussão sobre a resposta imune da expressão da agressividade intraespecífica frente a um estressor foi investigada em ratos. Aos 90 dias de vida, os animais foram divididos em três grupos: grupo controle (foram realizadas apenas as mensurações imunológicas); choque nas patas (FS) (os animais receberam FS individualmente) e grupo resposta agressiva intraespecífica (IAR) (os animais receberam FS e apresentaram IAR). Para as medições imunológicas, amostras de sangue foram coletadas imediatamente, 7 e 15 dias após FS ou IAR. O FS reduziu a quantidade total de leucócitos. Contudo, a agressividade, foi acompanhada, além da redução do número de leucócitos, por diminuição de linfócitos e aumento de neutrófilos. Ademais, foi observada também uma elevação no número de leucócitos associada a um aumento na resposta imune humoral uma semana após as IAR. Neste estudo, a expressão da agressividade intraespecífica frente a um estressor parece ativar o sistema imune e potencializar a resposta humoral antígeno específica.
INTRODUCTION
In several species, the aggressiveness is important for the survival, because the aggressive behavior guarantees access to food, reproduction, protection of the pups, struggle against the predators and defense of territory (Volvaka, 1995). On the other hand, the aggressiveness can take to traumas, wounds and also to higher susceptibility to the exposition to new diseases (Granger et al., 2000).
The hypothalamus-pituitary-adrenal axis (HPA) constitutes one of the fundamental elements of the mammalian adaptive response in most of the threatening situations (Castelnau & Lôo, 1993). The HPA axis and also other participating elements of the response to stress, as the sympathetic nervous system (Sgoifo et al., 1996; Lawrence & Kim, 2000), are components straightly associated to the expression